Alcohol use disorder is pervasive in society, imposing enormous socioeconomic costs and contributing to tens of thousands of deaths each year. Therefore, it is critical to identify mechanisms that influence risk for alcohol use disorderto improve prevention and treatment initiatives. Our proposal is a novel approach to studying the transmission of alcohol drinking behaviors across generations. While past research has focused on genes that modify risk for alcoholism, recent evidence has demonstrated that paternal experiences can impact offspring behavior via epigenetic mechanisms in sperm. We have shown that paternal preconception alcohol exposure in mice confers decreased preference for alcohol drinking, increased sensitivity to alcohol, and blunted stress responsivity phenotypes selectively to male offspring. The current study will investigate epigenetic mechanisms that drive these intergenerational alcohol- and stress-related behaviors. One mechanism recently implicated in epigenetic inheritance is transmission of small noncoding RNAs (smRNA) from sperm to the oocyte during fertilization. Thus, the first specific aim will test the hypothesis tha chronic alcohol exposure alters expression of smRNAs in sperm. Furthermore, we will directly test the hypothesis that sperm smRNAs mediate the intergenerational impact of paternal alcohol exposure by injecting sperm smRNA from alcohol-treated mice into fertilized embryos and characterizing the alcohol- and stress- related phentoypes in resulting offspring. The second aim will examine the relationship between stress and alcohol across generations. Stress is associated with alcohol abuse in humans and whether stress can influence intergenerational alcohol drinking is unknown. Therefore, we will employ a chronic variable stress model to test the hypothesis that paternal stress impacts alcohol-related behaviors in offspring. This project is an important step in elucidating the role of epigenetic inheritance in alcohol drinking behavior These studies are important because they examine causal epigenetic mechanisms that will provide novel insight into alternative modes of inheritance beyond the genome. The findings will advance our understanding of heritable risk factors that influence alcohol abuse with major implications for the development of novel treatment and prevention strategies.